Activation, overexpression, or gene amplification of EGF receptor, HER2 and HER3 are generally correlated with a poor breast cancer outcome. In contrast, HER4 is associated with good-prognosis breast cancers and growth inhibition. Also, in contrast to other EGF receptor family members, HER4 exhibits a unique cell biology, as it can be released from the membrane by proteolysis and the 80kDa soluble cytoplasmic tyrosine kinase is free to translocate within the cell (s80HER4). This HER4 cytoplasmic fragment has clearly evolved to work in the cell nucleus, as it contains three canonical nuclear localization and nuclear export sequences and, as our lab has found, functional cyclin-like D box sequences. We have demonstrated that s80HEFM is both necessary and sufficient to inhibit breast cancer cell line growth, including growth of HER2- overexpressing cells. Taken together, our findings suggest that nuclear HER4 can act in a tumor suppressor-like pathway. . One caveat is that HER4 can be alternately spliced, to one of two cytoplasmic domain isoforms, CYT1 and CYT2,. There is evidence that CYT1 and CYT2 may differ in their growth inhibition or proliferative signaling; our growth inhibition studies were performed with HER4 CYT1. Translations! Objective: To use cellular and animal models to elucidate differences in nuclear s80HER4 CYT1 and s80HERJf CYT2 action and to determine if HER4, HER4 nuclear localization, or specific HER4 isoforms correlate with breast cancer clinical characteristics or outcome. Aim 1: To determine if HER4CYT1 or s80HER4CYT1 has similar or different signaling capabilities and biological outcomes in breast cells as compared to HER4CYT2 and s80 2. We will use human breast cancer cells in culture and xenograft to immuno-compromised mice to examine the effects of each HER4 and s80 isoform on cell growth and tumor formation. We will use the HC11 cell line, derived from mammary gland of a mid-pregnancy Balb/C mouse, which retains the ability to undergo differentiation in culture, to examine the effects of each HER4 or s80HER4 isoform on mammary differentiation. Aim 2: To develop quantitative RT-PCR assays (real-time) for distinguishing and measuring HER4CYT1 and HER4CYT2 transcripts in clinical breast cancer specimens, as well as for measuring the expression levels of HER2, BRCA1, and Wwox. We will correlate RNAexpression with clinical characteristics and outcome in breast cancer samples. Aim 3: To correlate immunohistochemical data on the subcellular localization of HER4 isoforms with breast cancer and DCIS clinical characteristics and outcomes. We will develop antibodies to distinguish HER4CYT1 from HER4CYT2. We will use immunohistochemical staining to determine if HER4, nuclear localized s80HER4 HER4 isoform expression (CYT1 and CYT2) are associated with: i) breast cancer molecular subtype; ii) clinical outcome; or iii) stage and other pathologic characteristics of DCIS. Particular attention will be paid to invasive breast cancer/DCIS that co-express HER2 and HER4.